This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Amanda Napier has conducted supervised research training at the LFD since July, 2006. Her primary aims were to master biochemical techniques in order to carry out fluorescence lifetime imaging in fixed cells. Thus far she has done transformation procedures into DH5alpha cells in order to amplify DNA. The amplification of DNA procedures requires her to purify it from E.coli. She has followed the Qiagen protocol using miniprep spin columns which yields 9.5mg of plasmid DNA. She has amplified the yellow, cyan, yellow-cyan fluorescent protein constructs (CFP,YFP and CFP-YFP) sub-cloned into a mammalian expression plasmid. Once the DNA is purified (under the guidance of a postdoc), she transfected the DNA with lipofectamine into mammalian cells for expression. The cells are fixed with para-formaldehyde after 24hours of transfection and then mounted using a 1.7um thick coverslip. The cells are ready to image after 1 day. The lifetime imaging experiments will be conducted using the Berker-Hickel image acquisition card and the data will be analyzed using the phasor approach